HU224279B1 - Implant for repair of animal-cartilage, method for making the implant and cartilaginous tissue - Google Patents

Abstract

Field of the Invention The present invention relates to an animal cartilage (16) implant recovery implant (20) comprising a carrier matrix and cartilage cells attached to a carrier matrix, which is an animal-absorbing carrier matrix, and an implant (20) that can be introduced into the arthroscopic duct (26) of the implant (20), and the cartilage cells on a carrier matrix. are fixed. Another object of the present invention is to provide an implant for the invention comprising implant (20) embedded cartilage cells attached to the matrix and receiving host cell dust cells during the process; culturing cartilage cells on medium; providing a carrier matrix comprising a solid or semi-solid element to provide a basis for the growth of cartilage cells on it, and adding the cultured cartilage cells to the carrier matrix and making it possible to further cultivate the cartilage cells and to attach the cartilage cells; furthermore, an arthroscopic workpiece (26) is provided with an implantable implant (20), and the cartilage cells are further cultured on the carrier matrix and can be attached to the cartilage cells. Another object of the present invention is a cartilage cell implant comprising a live cartilage cell and a flexible carrier matrix, and apoptosis is attached to the carrier matrix, and a cell implant (26) can be implanted into the work channel (26) of the parenteral cell implant, and the cartilage cells are attached to the carrier matrix.

Description

The scope of the description is 24 pages (including 14 pages)

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The present invention relates to chondrocyte transplantation, cartilage implantation, healing, joint restoration, and prevention of arthritic conditions. The present invention relates to an implant for the restoration of animal cartilage by implantation, a method for making an implant and a cartilage implant.

In the United States, more than 500,000 joint plastic interventions and complete joint replacements are performed each year. There are approximately the same number of similar interventions in Europe. In Europe, this figure includes approximately 90,000 total knee replacement and approximately 50,000 interventions designed to heal knee injury. These numbers are essentially similar in the United States of America (PRAEMER, A. FURNÉR

5., RICE, D. P., Musculoskeletal Conditions in the United States, American Academy of Orthopedic Surgeons, Park Ridge, III., 1992, 125).

The most useful method would be to treat cartilage tissue regeneration and to do this early in the process of joint damage would reduce the number of patients who require surgical intervention for joint replacement surgery. Such a prophylactic treatment would also reduce the number of patients who develop osteoarthritis.

Techniques to repair cartilage surface usually attempt to induce cartilage repair using subchondral drilling, abrasion, or other methods that remove damaged cartilage and subchondral bone and leave the spongy bone-embedded, uninjured J.S. Clin. Orthop. 1974, 101, 61; FICAT RP et al., Clin. Orthop. 1979, 144, 74; JOHNSON LL, Operative Arthroscopy, McGINTY, JB Ed., Raven Press, New York, 1991, 341). .

COON and CAHN (Science 1966, 153, 1116) describe a technique for growing cartilage-producing cells from the spinal cord assembly of a chicken embryo. LATER, CAHN and LASHER (PNAS USA, 1967, 58, 1131) used the system to study the process of DNA synthesis as a prerequisite for cartilage differentiation. The cartilage cells reacted with growth to both EFG and FGF (GOSPODAROWICZ and MESCHER, J. Cell Physiology, 1977, 93), but eventually lost their ability to differentiate (BENYA et al., Cell, 1978, 15, 1313 ). A method for culturing cartilage cells has been described by BRITTBERG, M. et al., New Engl., J. Med.

1994, 331, 889) and is used with minor modifications. Cells cultured by these methods are used for autologous transplantation into the knee joint of patients. In addition, KOLETTAS et al. (J. Cell Science, 1995, 108, 1991) examined the expression of cartilage-specific molecules such as collagens and proteoglycans during long cell cultures. It was found that monolayer cultures (AULTHOUSE, A. et al., In vitro Cell Dev. Bioi., 1989, 25, 659, ARCHER, C. et al., J. Cell Sci. 1990, 97, 361;

HANSELMANN, H., et al., J. Cell Sci, 1994, 107, 17; BONAVENTURE, J., et al., Exp Cell Cell 1994, 212, 97) compared to suspension cultures grown on agar-based gels, alginate beads or shaken cultures (which maintain spherical cell morphology) as investigated by various scientists, in cultures, the chondrocytes do not change with the expressed markers, e.g. and IX. type collagens and large aggregating proteoglycans, aggrecan, versicane, and linker protein (KOLETTAS, E., et al., J. Cell Science, 1995, 108, 1991).

WAKITANI et al., Tissue Engineering, 4 (4), 429, 1989, describe the use of collagen type I in the treatment of cartilage injuries in animal experiments. In all cases, the greatest problem was the lack of biomechanical properties required for functional tissue repair.

Articular cartilage cells are exclusively specialized cells of embryonic origin (mesenchyma) found in cartilage tissue. Cartilage tissue is an avascular tissue whose physical properties depend on the extracellular matrix produced by the chondrocytes. During endocrine ossification, cartilage cells undergo a maturation process that leads to cellular hypertrophy, which is indicated by the onset of expression of type X collagen (UPHOLT, WB and OLSEN, R.R. “Cartilage Molecular”). Aspects, HALL, B. & NEWMAN, S. Eds., CRC Boca Raton 1991, 43; REICHENBERGER, E., et al., Dev. Bioi., 1991, 148, 562; KIRSCH, T. et al., Differentiation, 1992, 52, 89; STEPHENS, M. et al., J. Cell Sci., 1993, 103, 1111).

II. The high degree of degeneration of type I collagen in the outer layer of the joint surfaces of the joints is also caused by osteoarthritis. Meanwhile, the collagen matrix is weakened and subsequently fibrous, whereby matrix materials such as proteoglycans are depleted and eventually disappear. Fibrosis of debilitated osteoarthritis can penetrate all the way down to ossified cartilage and subcellular bone tissue (KEMPSON, GE et al., Biochem. Biophys. Acta, 1976, 428, 741; ROTH, V. and MOW, VC, J. Boné Joint 1980, 62A, 1102; WOO, SL-Y, et al., Handbook of Bioengineering, R. SCALES and S. CHIEN, ed., McGraw-Hill, New York, 1987, pp. 4.1-4.44).

WO 98/08469 discloses a method of making an implant containing cartilage cells fixed to a carrier matrix, and a method of treatment for improving the articular cartilage surface by implanting cartilage cells in a suitable matrix. In the latter process, an anti-haemorrhagic agent is applied to the site of injury, on which the carrier matrix is deposited with the cartilage cells and finally covered with a cover sheet. Cartilage cells are cultured in media containing 5-7.5% autologous serum at 37 ° C in CO ₂ medium.

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NEHRER, S. et al., Describe the in vitro culture of cartilage cells in two types of sponge-like matrix and the in vivo implantation of matrices with cartilage cells attached thereto; one of the matrices is type I bovine collagen and the other one is II. porcine collagen (Canine Chondrocytes Seeded in Type I and Type II Collagen Implants Investigated in Vitro, Journal of Biomedical Materials Research, 1997, Vol. 38, No. 2, pp. 95-104).

WO 97/46665 discloses a process for producing an implant for repairing damage to articular cartilage surfaces. The implant described herein contains cartilage cells arranged on a porous plate made of bone substitute material. The bone replacement material has a coarse side, which may be porous, reversibly deformable, and may be a bioabsorbable material, including type I collagen.

SITTINGER, M., et al., Describe a method for in vitro cartilage implantation by culturing cartilage cells on three-dimensional matrices. These matrices are made from polydioxanone and Vycryl polymers impregnated with bio-absorbable poly-L-lysine or II. type collagen (Engineering of Cartilage Tissue Using Bioreosorbable Polymer Carriers in Perfusion Culture, Biomaterials, GB, Elsevier Science Publishers BV, Barking, Vol. 15, No. 6, pp. 451-456).

U.S. Patent No. 5,736,372 discloses methods and artificial matrices for culture and implantation of cartilage cells.

EP 339 607 discloses a method for cartilage repair by implanting fibrinogen-containing, biodegradable, biocompatible, bioabsorbable immobilization carrier (BRIV) adhesive-embedded cartilage cells.

Basic developmental, histological, and microscopic autopsy descriptions of bone, cartilage, and other related tissues can be found, for example, in WHEATER, BURKITT, and DANIELS, Functional Histology, 2 ^nd Edition (Churcill Livingstone, London 1987, Chp. 4). A basic histological description of the lesions of bone, cartilage and other related tissues can be found in WHEATER, BURKITT, STEVENS and LOWE, Basic Histopathology (Churcill Livingstone, London, 1985, Chp. 21).

Although the necessity of cartilage transplantation is extensively discussed at least in the above-mentioned work, there remains a need for a satisfactory and effective procedure for resolving cartilage tissue repair by transplantation or otherwise. It is therefore an object of the present invention to provide an implant, a method for making it, and a cartilage implant, and a method for implanting the implant, as well as the devices and materials therefor.

On the one hand, the object is solved by the creation of an implant for the restoration of an animal cartilage by implantation comprising a carrier matrix and cartilage cells attached to the carrier matrix, a carrier matrix to be absorbed in the animal, and an implant into

On the other hand, the object is solved by providing a method for making an implant comprising cartilage cells attached to an implant carrier matrix, which process comprises recovering cartilage cells from a host unit; culturing the cartilage cells in culture medium; providing a carrier matrix comprising a solid or semi-solid element that provides a basis for growth of cartilage cells thereon; and adding the cultured cartilage cells to the carrier matrix, thereby allowing the culture of the cartilage cells to grow and the cartilage cells to fixate; and providing an implant that can be inserted into the arthroscope's canal and culturing the cartilage cells on the carrier matrix and allowing the cartilage cells to attach to it.

The object is further solved by providing a cartilage implant comprising living cartilage cells and a flexible carrier matrix, the cartilage cells being fixed to the carrier matrix, the cartilage implant being inserted into the arthroscopic work channel of the cartilage and the cartilage implant.

Thus, the implant includes a carrier matrix that provides the basis for cell growth and fixation. This implant can be implanted to restore the cells at the site of implantation. Optionally, the present invention provides a method of effectively treating cartilage tissue of an animal joint by implanting an implant containing cartilage cells fixed on an absorbable carrier matrix. In one embodiment, the carrier matrix is, for example, I or II. type I collagen and the cartilage cells are autologous or homologous cartilage cells.

The present invention makes it possible to treat an animal cartilage injury by implanting an implant at the site of cartilage injury by providing an implant containing cartilage cells attached to a carrier matrix to be absorbed in the animal and securing the implant at the site of cartilage injury. Preferably, the implant is secured to the site of injury with an adhesive or mechanical fastening device.

The present invention will now be described in detail with reference to the accompanying drawings, in which:

IA. A typical example of bone articulation is shown with a knee joint where the ends of the bones are covered by a cartilage joint surface.

IB. Fig. 6A shows a cartilage injury or cartilage damage on a cartilage joint surface at the arthritic end of a bone;

Figure 2 shows an implant according to an embodiment of the invention, a

Figure 3 shows the bending of the implant of Figure 2, which can then be inserted into an arthroscopic insertion device, such as that shown in Figure 4, for implantation;

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Figure 4 illustrates an insertion device for inserting an implant into the implant site,

Figure 5 schematically illustrates the placement of the implant of Figure 3 at the site of injury or damage to the cartilage joint using two inlet tubes that provide space for the arthroscopic devices,

Fig. 6 is a schematic sectional view showing a cartilage with non-intact cartilage injury or damage and an implant of the present invention fixed to the site of injury or damage;

Fig. 7 is a schematic sectional view showing a cartilage with non-intact cartilage injury or damage and an implant according to the invention fixed to the site of injury or damage by a mechanical fastening device;

Figure 8 is a drawing of an embodiment of a mechanical fastening device for securing an implant at the site of injury or damage;

Fig. 9 is a schematic sectional view showing a cartilage with injury or damage extending into the cartilage layer and an implant of the present invention fixed to the site of injury or damage;

Figure 10 is a schematic sectional view showing a cartilage with injury or damage extending into the cartilage layer and an implant according to the invention fixed to the site of injury or damage by a mechanical fastening device;

Figure A is a line drawing of a photomicrograph of a histological specimen showing a solid carrier matrix at the start of cartilage culture on it;

11A. Fig. 11A is a black and white copy of the photomicrograph mentioned in Fig. 11A, a

IIB. Fig. 11A is a line drawing of a photomicrograph showing the carrier matrix of Fig. 11A inoculated with cartilage cells after three weeks of culturing the cartilage cells;

11BB. 11B. a black-and-white copy of the photomicrograph of FIG

IIC. Fig. 6A is a photograph showing immunohistochemical staining of a carrier matrix formed from collagen with cartilage cells grown thereon and finally

IID. Fig. 3A is a photograph showing immunohistochemical staining of a collagen matrix with cartilage cells grown therein.

As mentioned above, the knee is the joint in the human body where cartilage injury or damage often occurs. 1A. Fig. 4A shows a typical joint end of a bone of a human knee joint. The knee joint 10 is made up of femur 12 and tibia 14 and healthy cartilage 16 covers the articular end of the femur 12. 1B. FIG. 4A shows an area of circular damage or injury 18 on the cartilage 16. FIG.

The present invention relates to a cartilage-restoring implant 20 which provides for the possibility of implantation using a device therefor. The implant 20 comprises a carrier matrix 22 and autologous or homologous cartilage cells 24 fixed therein.

Generally, the carrier matrix 22 is a substance that provides the basis for the growth of the cartilage cells 24 and is absorbed over time by the patient receiving the implant 20. Transplantation may be performed in a slightly invasive manner, by arthroscopic technique or by open surgery. The method of the invention involves the use of appropriate cartilage cells 24 from both pedigree (allogeneic) and alien species (xenogeneic) in the repair of cartilage 18 damage.

Figure 2 shows such an implant 20. More specifically, the implant 20 contains the carrier matrix 22 together with the attached cartilage cells 24. The carrier matrix 22 may be solid or gel-like, having sufficient spatial structure to maintain a stable shape over an appropriate period of time that allows the cartilage cells to grow on it both before and after transplantation, and that provide a system that is similar to their natural environment for the cartilage cells, thereby optimizing the growth and differentiation of the cartilage cells.

The carrier matrix 22 remains stable for a period of time sufficient to completely regenerate the cartilage and is then absorbed by the body for a period of time, such as two or three months, without leaving significant traces and without producing toxic degradation products. Who. The term "absorption" is used to include any process whereby carrier matrix 22 is degraded by natural biological processes, and degraded carrier matrix and degradation products are cleared, such as through lymphatic vessels and blood vessels. Accordingly, the carrier matrix 22 is preferably a non-antigenic membrane material that is absorbed by physiological processes. Further, the carrier matrix 22 preferably forms a sheet shape with a relatively smooth surface 21 and a relatively rough surface 23. For example, the rough surface 23 is fibrous and generally faces the lesion 18 of the cartilage 16 and promotes the growth of cartilage cells inwardly, while the smooth surface 21 generally does not face the lesion 18 of the cartilage 16 and is resistant to tissue penetration.

In one embodiment, the carrier matrix 22 is composed of polypeptides or proteins. Preferably, the polypeptides or proteins are derived from natural sources, such as mammals. However, it is mes4

Chemical substances whose physical and chemical properties are comparable to those of naturally occurring polypeptides or proteins can also be used to form the carrier matrix 22. It is also advantageous if the carrier matrix 22 is reversibly deformable, for example, when the user is working with the implant 20 and if, after manipulation, it regains its original shape, as described below, in accordance with one object of the invention.

One of the preferred materials for forming the carrier matrix 22 is collagen, which can be obtained, for example, from horses, pigs, cattle, sheep and chickens. Suitable materials for forming the carrier matrix 22 include Chondro-Cell (a commercially available type II collagen matrix available from Ed. Geistlich Söhne, Switzerland) and Chondro-Gide (a commercially available type I collagen matrix). available from Ed. Geistlich Söhne, Switzerland). The carrier matrix 22 formed from collagen type I material is slightly stiffer than that of matrix II. Type 22 carrier matrix, but all. The carrier matrix 22 formed from type I collagen may also be used.

The implant 20 described above may be formed, for example, by culturing cartilage cells 24 on the carrier matrix 22 as described in more detail below.

To prepare an autologous implant, a cartilage biopsy is first obtained by arthroscopic technique from a non-strain part of a patient's joint and delivered to the laboratory in culture medium containing 20% fetal calf serum. The cartilage biopsy is then treated with an enzyme, such as trypsin-ethylenediaminetetraacetic acid (EDTA), a protein-degrading enzyme and binder, to separate and extract cartilage cells. The extracted cartilage cells are then cultured on the culture medium, with an initial cell count of about 50,000 and at the end of culturing with about 20 million or more.

Three (3) days prior to replanting, the culture medium was changed to a transplant medium containing 10% autologous serum (i.e., serum extracted from the patient's blood as described below). The cultured cartilage cells in the transplantation medium are then soaked in, penetrated into, and continued to divide with the carrier matrix 22 to form the implant 20. The implant 20 is then implanted at the site of injury 18 on the patient's cartilage.

It has been found that the lesion 18 can be either treated directly, slightly enlarged, or surgically shaped prior to implantation to allow space for the implant 20. The following is an example of the culture process, as well as the culture medium and the transplantation medium, starting with a laboratory procedure for the treatment of the recovered cartilage biopsy and a description of the culture of the cartilage cells.

The culture medium (hereinafter referred to as the "culture medium") used for the treatment of cartilage biopsy and growth of cartilage cells during the culture process is prepared as follows. Mix 2.5 ml (70 micromol / l) gentomycin sulfate, 4 ml (2.2 micromol / l) amphotericin (marketed as Fungizone, available from Squibb), 15 ml (300 micromol / l) 1 ascorbic acid, 100 ml (20% final concentration) of fetal bovine serum, and the remaining DMEM / F12 medium to produce approximately 400 ml of culture medium. (The same culture medium is used to transport cartilage biopsy from hospital to laboratory as in which the cartilage cells are extracted and in which they divide.)

The patient's blood is centrifuged at about 3000 rpm to separate the serum (serum) from the other blood components. The separated serum is retained and used at a later stage of the culture or transplantation process.

For autologous transplantation, the cartilage biopsy previously obtained from the patient is transported to the laboratory in the culture medium described, where the culture is performed. The culture medium is filtered to separate the cartilage biopsy and upon arrival at the laboratory, the former is destroyed. The cartilage biopsy is then washed at least three times in pure DMEM / F12 to remove the film coat of fetal calf serum from the cartilage biopsy.

The cartilage biopsy is then washed in a preparation prepared by adding 28 ml (0.055 concentration) trypsin EDTA to the culture medium described above. In this formulation, incubate for 5 minutes at 37 ° C and 5% CO ₂ . After incubation, the cartilage biopsy is washed two or three times in culture medium to completely purify the biopsy of trypsin enzyme. The cartilage mass is then weighed. Typically, the smallest amount of cartilage required for culturing cartilage cells is about 80-100 mg. A slightly larger amount, such as 200-300 mg, is preferred. After the measurement, the cartilage is placed in a mixture of 2 ml (5000 enzyme units) of collagenase (degrading enzyme) and approximately 50 ml of pure DMEM / F12 medium and then milled to allow the enzyme to partially degrade the cartilage. After grinding, transfer the milled cartilage to a flask using a funnel and add about 50 ml of a mixture of collagenase and pure DMEM / F12 to the flask. The milled cartilage is then incubated for 17-21 hours at 37 ° C and 5% CO ₂ .

In one embodiment, the incubated minced cartilage is then screened through a 40 µm mesh screen and centrifuged (at 1054 rpm or 200 times gravity) for 10 minutes and then washed twice in culture medium. The cartilage cells are then counted to determine their viability, and the cartilage cells are incubated at 37 ° C and 5% CO ₂ for at least two weeks, during which time the culture medium is changed three or four times.

At least three days prior to transplantation into the patient, the cartilage cells were treated with trypsin and

The supernatant is removed from the culture medium by centrifugation and transferred to a transplant medium containing 1.25 ml (70 micromol / l) gentomycin sulfate, 2 ml (2.2 micromol / l) amphotericin (Fungizone brand, Squibb). antifungal agent available), 7.5 ml (300 micromol / l) of 1-ascorbic acid, 25 ml (10% final concentration) of autologous serum and the remaining DMEM / F12 medium to form approximately 300 ml of transplantation medium.

The carrier matrix 22 is then cut to fit at the bottom of a well of a NUNCLON cell culture plate and then aseptically placed at the bottom of the well with 1-2 ml of transplantation medium. An appropriate number of cultured cartilage cells (e.g., 3 to 10 million cartilage cells) in about 5 to 10 ml of transplantation medium are then digested with the carrier matrix 22 and incubated for about 72 hours at 37 ° C with 5% CO ₂ to continue to grow. During this incubation, the cartilage cells settle in colonies and attach to the carrier matrix 22. It has now been found that the carrier matrix 22 provides a basis for the growth of the cartilage cells and that the cartilage cells are sufficiently fixed to form the implant 20 without significantly losing the biomechanical properties of the carrier matrix 22. The carrier matrix 22 also provides a favorable environment for the growth of cartilage cells after the implant 20 has been implanted at the site of injury to the cartilage 16.

In another embodiment, after the 17-21 hour incubation period and after the cell number and viability assays discussed above, the cartilage cells are transferred to the transplant medium and the cartilage cells grow directly on the carrier matrix for at least two weeks.

It has been found that the implant 20 is temporarily deformable without being mechanically damaged or that the cartilage cells attached to the carrier matrix 22 are lost. This deformation is completely reversed as soon as the implant 20 is inserted into the joint or placed on the surface to be treated, as will be described later.

Accordingly, in accordance with other objects of the present invention, the carrier matrix 22 on which the cartilage cells have grown in sufficient number or on which the cartilage cells have been deposited in an appropriate number is temporarily deformable so that it can be placed in an arthroscope tool without mechanical or mechanical damage. the cartilage cells on it would be lost.

However, it has been found that the carrier matrix 22 can be fixed to the site of injury to the cartilage 16 by glue or mechanical fixation without adversely affecting the in situ differentiation of the cartilage cells and the regeneration of the natural cartilage tissue.

Implantation requires a means by which the implant 20 can be delivered to the implant site and a mechanical fastening device which holds the implant 20 at the implant site.

In one embodiment of the implantation procedure, arthroscopic technique is used. Figure 3 illustrates how the implant 20 can be wrapped around its diameter to obtain a spirally screwed cylindrical implant 20, thereby delivering the implant 20 through the working channel 26 of the arthroscopic insertion device 28 to the implant site. A suitable arthroscopic insertion device 30 is shown in Figure 4.

The arthroscopic insertion device 30 shown in FIG. 4 comprises a working passage 32 having a diameter and length that allows it to penetrate into the joint in question and deliver an implant of the desired size. For example, in most cases, the passage 32 has a diameter of about 8-20 mm and a length of about 30-60 cm. Inside the working passage 32 is a longitudinally movable inlet passage 34 having a retractable and removable needle 36. The needle 36 extends over the entire length of the inlet duct 34 and provides a means for delivering fluids there to the implant site. Within the passage 32, the inlet passage 34 may be moved by a telescopically movable handle 38.

The lead-in device 30 also includes a bell 40 made of rubber or other suitable material which is slidably secured to the lead-in device 30. During use, the bell 40 encircles the site of injury to the cartilage 16, thereby excluding fluids, such as blood and other natural fluids, that cannot flow to the site of injury to the cartilage 16. The insertion device 30 further has two or more outwardly biased gripping means 42 attached to the handle 38, which grip the implant 20, insert it, and place it at the site of implantation. During use, as the handle 38 is telescopically moved toward or away from the user, the gripping members 42 slide into the inside of the working passage 32 and move toward each other, thereby engaging (as the handle 38 is moved toward the user) to release the catch (as soon as the handle 38 is removed from the user). Such telescopic motion may be controlled (not shown) by an actuator located within the handle 38, which allows the inlet channel 34 and the grip elements 42 to be extended and retracted within the working channel 32.

5-7. Figures 3 to 5 show a typical arthroscopic procedure in which the implant 20 is implanted at the site of implantation, for example in the knee joint. The damaged cartilage affected by injury 18 is removed from the site of injury 18, preferably at a depth that does not touch the subchondral layer 44, thereby creating a recess 46 (see Figures 6 and 7). After the lesion 18 of the cartilage 16 has been removed, the site of the lesion 18 is prepared for receiving the implant 20. If 44 layers below the cartilage are covered6

EN 224 279 Β1 to the extent that bleeding occurs at the site of implantation, the site must first be covered with an absorbent material that acts as a hemostatic.

On the other hand, site preparation may include providing biocompatible adhesive to needle 36 via needle 36. Such a biocompatible adhesive, shown as adhesive 48 in Figure 6, may include an organic fibrin glue (e.g. Tisseel fibrin-based glue, Baxter, Austria) or a fibrin glue prepared from an autologous blood sample in a surgical operating room.

The implant 20 is pre-cut to the desired size and twisted as shown in FIG. 5 to take the spiral cylindrical shape, then gripped by the grips 42 and held at the end of the arthroscopic insertion device 30. The arthroscopic insertion device 30, which holds the implant 20 at its end, is then inserted into the implant site via an inlet tube 33, the implant 20 is released from the grip of the grips 42 and unwound with the grips 42, or left to unwind. working channel. The inlet tube 33 is comprised of one or more channels that allow devices such as the inlet and imaging devices to be introduced into the site of implantation. Using the gripping elements 42, the implant 20 is manipulated so that the rough surface 23 of the implant 20 faces the recess 46 and is held gently in the recess 46, thereby allowing the adhesive 48 to solidify and bind the implant 20 to the recess 46. recess.

In another embodiment (Fig. 7), mechanical fastening means, such as absorbable nails, rivets, screws or seams, are used to secure the implant 20 in the recess 46. Suitable nails include Ortho-Pin, a commercially available lactide copolymer nail (Ed. Geistlich Söhne, Switzerland). Figure 8 illustrates an embodiment of a resorbable nail 50. In this embodiment, the nail 50 comprises a head portion 52, an intramedullary channel 54 located within the stem portion 56, and one or more mounting flanges 58. The dimensions of the nail 50 may vary with the particular application, but typically the nail 50 is about 10-15 mm long, the diameter of the head 52 is about 4 mm, the diameter of the intramedullary canal 54 is about 1.2 mm, and the stem 56 is about 2 mm the mounting flange 58 has a diameter of about 2.5 mm. The clamping flange 58 serves to secure the nail 50 to the healthy cartilage tissue surrounding the lesion 18 of the cartilage 16. The nail 50 may be formed of any material that is not a hazard to the body and that is absorbed or degraded by the body over a period of time. For example, the nail 50 may be made of polylactide.

However, the adhesive 48 and the mechanical fastening means, such as nails 50, can be used together to fix the implant 20 of the present invention in the recess 46.

As shown in FIG. 6, a second inlet tube 60 including one or more channels may also be used to provide devices at the implant site that assist in positioning the implant 20, adhesive and / or mechanical fixation device, or providing imaging means. placement at the implantation site. Such a separate inlet tube 60 may also be used to perform the activities described for the arthroscopic inlet device 30 or other arthroscopic device.

As mentioned above, in cases where the lesion 18 of the cartilage 16 reaches or transcends the sub-cartilage 44, or when the lesion 18 requires removal of the cartilage along with the removal of the sub-cartilage 44 or deeper layer than the 9 10 and 10, the above procedure should be modified to include the placement of the haemostatic agent 62 in the well 46 before the implant 20 is placed. Bleeding agent 62 prevents the formation of blood vessels, osteocytes, fibroplasts, etc. grow or proliferate so that they also penetrate the resulting cartilage tissue. In our opinion, this provides an opportunity for glass cartilage (hyaline cartilage) to grow at the transplant site. A suitable haemostatic agent 62 prevents vascularization and proliferation of the affected cartilage tissue, thereby favoring the development of cartilage tissue and the development of full thickness of the cartilage at the site of injury.

Preferably, the haemostatic agent 62 remains stable for an extended period of time, thereby allowing the cartilage tissue to be completely regenerated and subsequently absorbed or otherwise degraded by the body over a period of time. A suitable haemostatic agent is Surgicel W1912 (Ethicon, Ltd., United Kingdom), an absorbable haemostatic agent made from oxidized, regenerated, sterile cellulose.

The surgical instruments described above may be made of any material, such as metal and / or plastic or silicone, suitable for the manufacture of disposable or reusable surgical instruments.

Certain aspects of the invention have been investigated in vitro in order to study the behavior of cartilage cells in contact with different types of carrier matrices. Based on these in vitro experiments, the mechanical resistance of various materials during the arthroscopic procedure can be predicted and the experiments also provide information on the growth of cartilage cells.

Parts of the invention which correspond to these and other objects will be more readily understood by reference to the following examples, which are provided by way of illustration only, and are not intended to limit the scope thereof.

Example 1

For three weeks, the culture medium described above was cultured in CO ₂ incubator at 37 ° C for Verigen Translplantation

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Service ApS (Copenhagen, Denmark) or the 100th purity laboratory of the University of Lübeck (Lübeck, Germany). (Note that other culture media may also be used for cartilage culture.) Cells were treated with trypsin-EDTA for 5 to 10 minutes and counted using a Trypan Blue staining procedure in a Bürker-Turk chamber. The cell number was adjusted to 7.5-10 ⁵ cartilage cells per milliliter. A NUNCLON plate was left uncovered in a grade 100 laboratory.

The carrier matrix material, more particularly Chondro-Gide collagen membrane, was cut to size to fit the bottom of a well of a NUNCLON cell culture plate. In this case, a circular piece about 4 cm in diameter was placed under aseptic conditions at the bottom of the well.

After three weeks, the cartilage cells were transferred from the culture medium to the transplant medium described above, and approximately 5 to ⁶ cartilage cells were placed directly on top of the carrier matrix in 5 ml transplantation medium. The plate was incubated for three days at 37 ° C in a CO ₂ incubator. Thereafter, the cartilage cells settled in colonies and began to grow on the carrier matrix and could not be removed from the carrier matrix by rinsing with the medium or even weak mechanical force on the matrix.

After the incubation period, the transplantation medium was filtered and the carrier matrix, together with the grafted cartilage cells, cooled in 2.5% glutaraldehyde to which 0.1 M sodium dimethyl-arsenic acid was added as a fixative. The carrier matrix was stained with Safranin O for histological examination. A line drawing of the micrograph taken from this is shown in Figure 11A. Black and white copy 11AA. As shown in Figures 4 to 5, this can be further studied by micrograph.

Example 2

For three weeks, the culture medium described above was cultured in a CO ₂ incubator at 37 ° C in a purity class 100 laboratory of the Verigen Translplantation Service ApS (Copenhagen, Denmark) or the University of Lübeck (Lübeck, Germany). Cells were trypsinized for 5 to 10 minutes using trypsin-EDTA and counted in a Bürker-Turk chamber using a Trypan Blue staining procedure. The cell number was adjusted to 7.5-10 ⁵ cartilage cells per milliliter. A NUNCLON plate was left uncovered in a grade 100 laboratory.

The Chondro-Gide carrier matrix, as in Example 1, was cut to the proper size to fit the bottom of a well of a NUNCLON cell culture plate. In this case, a circular piece about 4 cm in diameter was placed under aseptic conditions at the bottom of the well.

After three weeks, the cartilage cells were transferred from the culture medium to the transplant medium described above, and approximately ^{5 to 5} cartilage cells were placed directly on top of the carrier matrix in 5 ml transplant medium and plated on the surface of the carrier matrix.

The plate was incubated for three weeks at 37 ° C in a CO ₂ incubator.

After the incubation period, the transplant medium was filtered and the carrier matrix, together with the grafted cartilage cells, cooled in 2.5% glutaraldehyde, to which 0.1 M sodium dimethyl-arsenic acid was added as a fixative. The carrier matrix was stained with Safranin O for histological examination. For immunohistochemistry, the collagen membrane was fixed in methanol / acetone and rabbit antihuman II. type collagen and mouse anti-human aggrecan using aggrecan and type II. type collagen was stained. Primary antibodies were visualized using fluorescent secondary antibodies. 11B is a line drawing of a photomicrograph taken therefrom. Fig. 4A shows the cartilage cells 24. Black and white copy 11BB. As shown in Figures 4 to 5, this can be further studied by micrograph.

During the three-week incubation period on the Chondro-Gide carrier matrix, it was observed that the cartilage cells grew and proliferated on the carrier matrix, forming colonies in the middle of the carrier and covering the surface.

Example 3

For three weeks, the culture medium described above was cultured in a CO ₂ incubator at 37 ° C in a purity class 100 laboratory of the Verigen Translplantation Service ApS (Copenhagen, Denmark) or the University of Lübeck (Lübeck, Germany). Cartilage cells were treated with trypsin for 5 to 10 minutes using trypsin-EDTA and counted in a Bürker-Turk chamber using a Trypan Blue staining procedure. The cell number was adjusted to 7.5Ί0 ⁵ cartilage cells per milliliter. A NUNCLON plate was left uncovered in a grade 100 laboratory.

The Chondro-Gide carrier matrix, as in Example 1, was cut to the proper size to fit the bottom of a well of a NUNCLON cell culture plate. In this case, a circular piece about 4 cm in diameter was placed under aseptic conditions at the bottom of the well.

After three weeks, the cartilage cells were transferred from the culture medium to the transplant medium described above, and approximately 5 to ⁶ cartilage cells were placed directly on top of the carrier matrix in 5 ml of transplant medium and plated on the surface of the carrier matrix. The plate was incubated for three weeks at 37 ° C in a CO ₂ incubator.

The carrier matrix was then incubated with collagenase for 16 hours together with the grafted cartilage cells. The carrier matrix supporting the cartilage cells was then centrifuged. Cells were then inoculated onto a NUNCLON plate and aliquots of cells were counted in a Bürker-Turk chamber for determination of viability using the Trypan Blue staining procedure.

HU 224 279 Β1

The micrograph of this is shown in Fig. 11C. is shown. The calculated total cell number was found to be 6Ί0 ⁶ and the viability was> 95%.

Example 4

Animal experiments were carried out at the facilities of the University of Lübeck, Germany.

Four rounded cartilage lesions of 7 mm in diameter were created in the cartilage of the knee joints of two sheep. All interventions iv. Ketanest / Rompun was performed under full anesthesia. The lesions were created by drilling two holes in the cartilage bearing the medial femur of the femur (femur medial condilus) and two holes in the femuropatellar and tibiofemural cartilages. In each of these two areas, one of the two holes extended beyond the cartilage and subterranean cartilage to the bone, while in each area the other hole did not penetrate the cartilage to the cartilage border.

At the same time, a cartilage sample was obtained from the non-load bearing portion of the sheep's knee joint.

From this cartilage sample, cartilage cells were prepared on a carrier matrix as in Example 3 over a period of six weeks.

Cartilage cells on the Chondro-Gide carrier matrix were implanted by arthroscopic surgery. For one sheep, the matrix was fixed with fibrin glue to the treated area, while for the other sheep, the matrix was fixed with the polylactide nails described in connection with the present invention.

The sheep were kept separate and the knee joint was properly secured for one week.

The sheep were then allowed to move freely. Examination of the joint showed healing of the injury; that the cell-bearing matrix implant was fixed at the site of cartilage injury and that cartilage tissue was regenerated at the site of cartilage injury.

Although the above discussion relates in part to a method of growing cartilage cells on a carrier matrix in a glass vessel such as a NUNCLON plate and replacing the culture or transplant medium for proper cell culture, the present invention also relates to a method of bioreactor, such as Model 1302, available from MinuCells GmbH Ltd. (D-93077, Bad Abbach, Germany). When using a bioreactor, a constant flow of culture medium or culture medium passes along the carrier matrix and the cartilage cells can grow at a higher rate on the carrier matrix without having to change the culture medium or transplant medium every 24 hours as in the case of NUNCLON plates. . It is known that the use of such a bioreactor will result in cartilage cells growing obliquely as a result of the flow of the culture or transplantation medium through the bioreactor. 11D. Fig. 4A is a photomicrograph of cartilage cells grown on a carrier matrix in a bioreactor.

The cartilage cells can be cultured either completely in the glass vessel or on the carrier matrix throughout the culture process or in the transplant medium throughout the culture process. That is, there is no need to transfer the cartilage cells from the culture medium to the transplantation medium. The cartilage cells may be transferred from the culture medium to the transplant medium, or vice versa, at any time during the culture process, depending on the actual status of the cartilage cells, the degree of cartilage growth, or the condition of the patient. Cartilage cells, whether on culture media or transplantation media, should be immersed in the carrier matrix only 2-3 hours prior to transplantation to allow sufficient number of cartilage cells to attach to the carrier matrix.

It has been observed that, if a bioreactor is not used, the culture or transplantation medium currently being used in the current phase of the cell culture process should be changed, for example, at about 24 to 96 hours, for example depending on cell number or cell viability.

Although the invention has been described with reference to specific embodiments and embodiments, the invention is not limited thereto. In the most general sense, the present invention includes any cartilage implant (or its use) that is preferably flexible, and preferably contains a carrier matrix that provides a base for living cells to be absorbed in the living organism, and to grow and bind to the carrier matrix for a minimum period. This attachment can be accomplished by the penetration of cells into the matrix surface. Preferably, the carrier matrix also provides the implant with sufficient physical integrity to allow manipulation of the implant, such as that required during implantation into a living organism.

The appended claims are intended to cover not only the embodiments described, but also all the various embodiments, variations and equivalent solutions which may be practiced by one of ordinary skill in the art; these embodiments, variants, and equivalent solutions are also within the scope of the invention and are within the scope of the claims.

Claims (28)

PATENT CLAIMS An implant for recovering an animal cartilage by implantation comprising a carrier matrix (22) and cartilage cells (24) attached to the carrier matrix (22), the carrier matrix (22) being an arthroscope of the implant (20). an implant (20) can be inserted into the work channel (32), and the cartilage cells (24) are fixed on the carrier matrix (22). An implant according to claim 1, characterized in that the carrier matrix (22) is a flat element that provides the basis for the growth of cartilage cells (24), which provides the implant (20) with physical integrity and allows manipulation. The implant of claim 1, wherein the carrier matrix (22) comprises polypeptides or proteins. Implant according to claim 1, characterized in that the carrier matrix (22) is made of collagen. The implant of claim 4, wherein the collagen is a collagen selected from the group consisting primarily of equine, porcine, bovine, sheep and chicken collagen. 6. The implant of claim 4, wherein the collagen is porcine collagen. The implant of claim 1, wherein the carrier matrix (22) is solid. The implant of claim 1, wherein the carrier matrix (22) is gel-like. The implant of claim 4, wherein the collagen is type I collagen. 10. The implant of claim 4, wherein the collagen II. type collagen. The implant of claim 1, wherein the implant (20) is reversibly deformable. The implant of claim 1, wherein the carrier matrix (22) has a rough surface (23). The implant of claim 12, wherein the rough surface (23) is porous. The implant according to claim 1, characterized in that the carrier matrix (22) has a smooth surface (21). Implant according to claim 1, characterized in that the carrier matrix (22) has a rough surface (23) and a smooth surface (21). A method of making an implant (20) comprising cartilage cells (24) attached to an implant (20) carrier matrix (22), the method comprising: (a) recovering cartilage cells (24) from a host unit; (b) culturing the cartilage cells (24) in the medium; (c) providing a carrier matrix (22) containing a solid or semi-solid element that provides a basis for growth of the cartilage cells (24); and (d) adding the cultured cartilage cells (24) to the carrier matrix (22), thereby allowing the cultured cartilage cells (24) to be further cultured and the cartilage cells (24) to attach; characterized in that an implant (20) can be inserted into the arthroscopic work channel (32), and the cartilage cells (24) are further cultured on the carrier matrix (22) and the cartilage cells (24) are allowed to attach therein. The method of claim 16, wherein the sheet-like carrier matrix (22) is used. The method of claim 16, wherein the carrier matrix (22) comprises polypeptides or proteins. The method of claim 16, wherein the collagen carrier matrix (22) is used. 20. The method of claim 19, wherein the carrier matrix (22) is selected from the group consisting of collagen, porcine, bovine, ovine and chicken collagen. The method of claim 19, wherein the porcine collagen carrier matrix (22) is used. The method of claim 19, wherein the type I collagen carrier matrix (22) is used. 23. The method of claim 19, wherein type collagen carrier matrix (22). The method of claim 16, wherein the solid support matrix (22) is used. The method of claim 16, wherein the gel-like carrier matrix (22) is used. The method of claim 16, wherein the absorbent carrier matrix (22) is used. A cartilage implant comprising live cartilage cells (24) and a flexible carrier matrix (22), and the cartilage cells (24) are fixed to the carrier matrix (22), characterized in that the cartilage cell implant (32) can be inserted into the arthroscopic 24) are fixed on the carrier matrix (22). A cartilage implant according to claim 27, characterized in that the carrier matrix (22) is capable of being absorbed into the animal by natural biological processes in the animal.